An immobilized-enzyme system for the determination of sialic acid using cloned neuraminidase from Clostridium perfringens A.T.C.C. 10543
Chien, C.H.; Wei, Y.H.; Yeh, S.F.; Li, C.P.
Biotechnology and Applied Biochemistry 19(1): 51-60
1994
ISSN/ISBN: 0885-4513 PMID: 8136081 Document Number: 433346
Neuraminidase expressed by cloned nanH of Clostridium perfringens has been immobilized and employed to determine the concentration of sialic acid in human serum. Two enzyme pairs, cloned neuraminidase-N-acetylneuraminate (NANA) lyase and pyruvate oxidase-peroxidase, have been respectively coimmobilized on to 1,12-aminododecane-agarose with glutaraldehyde. The relative specific activities of the co-immobilized neuraminidase and NANA lyase were 61 and 77%, and those of pyruvate oxidase and peroxidase were 51 and 96% of the corresponding soluble enzymes respectively. The optimal reaction pH at 37 degree C for each of the co-immobilized enzymes was about 1 pH unit higher than that of the corresponding soluble enzyme. The optimal reaction temperature of peroxidase was increased as a result of immobilization. The thermostability of the immobilized cloned neuraminidase, NANA lyase, pyruvate oxidase and peroxidase were increased 80-, 83-, 115- and 147-fold at 45 degree C over the soluble forms respectively. The results correlated satisfactorily with those obtained by using a soluble enzyme system. The system is thus a reliable assay method for sialic acid in serum.